Inhibition of complement activation and complement modulation by use of modified oligonucleotides

ABSTRACT

Oligomeric compounds are described wherein said compounds comprise modified oligonucleotides (P=S) which modulate complement activity. Methods and processes for the uses of such oligomeric compounds are also described. The oligomeric compounds may be used therapeutically to modulate complement activity in order to inhibit undesirable complement mediated events, such as for example, to treat inflammation, and/or to activate complement.

FIELD OF THE INVENTION

This invention concerns methods for the inhibition and/or modulation ofthe complement mediated immune response using synthetic nucleic acidmolecules. The nucleic acid molecules may be synthetic nucleic acidmolecules, such as oligonucleotides, wherein at least one of the esterlinkage moieties of the oligonucleotide is replaced with a thioatelinkage, such as in for example phosphorothioates. The methods describedherein are useful as therapies for treating abnormal and/or undesirableconditions which can arise as a result of complement activation. Furtheruses as diagnostics and research reagents are also included in thepresent invention.

BACKGROUND OF THE INVENTION

The complement system is an important means by which a host defendsitself against infection. The complement cascade system is a componentof the immune system that helps provide a natural immunity againstinvading microbes and is also an effector arm of antibody mediatedhumoral immunity. Complement is responsible for activating cells andother molecules involved in the inflammatory process as well as beingdirectly related to the destruction of microbial invaders. Theactivation of complement involves a cascade of proteolytic reactionsthat lead to the release of inflammatory mediators and result in theassembly of the microbial membrane attack complexes which, in turn, lyseinvading microbial cells. This cascade system has been characterized ascontaining at least thirty serum and membrane proteins that areactivated by antibody-antigen complexes or by the invasion, in a host orexperimentally in culture, by a microorganism, or other antigenicmolecules. Complement proteins may be grouped into three generalcategories: activating components, receptors, and positive and negativeregulators.

The complement cascade consists of two major branches, the classical andalternative pathways. Though these pathways are initiated differently,they converge at the step of complement protein C3 activation (see FIG.5). The complement cascade can mediate undesirable cellular damage ininflammatory, immune or autoimmune (auto-antibody-mediated) conditionssuch as; myasthenia gravis, immune complex excess syndromes such assystemic lupus erythematosus, ischemia-reperfusion states, hyper-acuterejection of transplants, organ failure conditions such as adultrespiratory distress syndrome, Alzheimer's disease and relatedneurodegenerative disorders, among others.

A series of regulatory proteins are involved in the control of thecomplement cascade. These proteins are considered part of the complementsystem and act to block endogenous complement activity at eitherinitiation or the formation of the membrane attack complex. varioustherapeutic agents are being developed that block different steps in thecomplement cascade.

Complement is a group of serum proenzymes that are activated by antigenbound immunoglobulin or by membrane components on gram negative bacteriaor fungi. The alternative pathway of the complement system isinitialized by either the introduction of an endotoxin such aslipopolysaccharide [LPS], a component of the cell walls of gram negativebacteria or for instance by zymosan, a component of yeast cell walls, orby aggregated IgA.

The classical pathway of the complement system is initialized byComplement protein C1 binding to antigen bound IgG or IgM. Both pathwaysconverge at the formation of C3 convertase at which point anamplification takes place that generates literally thousands of C3a andC3b fragments. C3b fragments can bind to complement protein complexC4b2a to form C4b2a C3b which is called C5 convertase and generatesthousands of C5a and C5b fragments. C3b can also be used to regenerateC3 convertase which causes a greater amplification of complement proteinsplit product C3a. Split products C3a and C5a interact with receptors onmast cells to cause them to release histamine. Histamine inducesinflammation which is generally considered protective, but in conditionscharacterized by improper complement activation and/or regulationinflammation can lead to damaged tissue.

One approach to inhibit complement mediated effects is by depletingcomplement. Depleting complement involves reducing the proteinsresponsible for the regeneration of C3 or C5 convertase and therebyreducing the amount of C3a and C5a produced. In this way complement isdepleted or “used up”. One such method for depleting complementcomponent C3 convertase involves allowing C3 convertase to form and thenbinding split product C3b in order to reduce the further amplificationof C3 convertase formation which can lead to C5 convertase formation.

In another approach for inhibiting complement the pathway is inhibitedbefore the formation of C3 convertase. Inhibition of the formation of C3convertase limits the production of split products C3b and C3a andfurther limits the formation of C5 convertase. Using this approachcomplement activation is blocked rather than depleted.

Candinas et al., describe the activation and depletion of complement byusing cobra venom factor in conjunction with a recombinant solublecomplement receptor type 1 protein (sCR1), and the use of such moleculesin treating hyperacute xenograft rejection. (Candinas, D. et al.,Transplantation 1996 15;62(3):336-342) sCR1 is a recombinant proteinthat has been shown to inhibit both the classical and alternativepathways of complement and thereby limits the production ofproinflammatory products such as the anaphylatoxins (complement proteinsC3a, C4a and C5a). sCR1 has also been described by Moore, F D Jr., asthe first protein useful to treat adverse clinical situations which arecomplement-dependent, and further describes potential uses for sCR1 totreat thermal injury, ARDS, septic shock, and ischaemia/reperfusioninjury events such as myocardial infarction after thrombolytic therapy.(Moore, F D Jr., Adv. Immunol 1994 56:267-299) U.S. Pat. No. 5,856,297,Fearon et al., issued Jan. 5, 1999 claims pharmaceutical compositionscomprising a CR1 protein in various modifications and describes CR1 andthe recombinant forms of the protein as being useful in the diagnosisand treatment of disorders involving complement activity andinflammation.

Other proteins have been investigated for their usefulness in inhibitingor modulating complement. For instance, Human IgG has been used tobalance complement activation in a pig-to-primate cardiacxenotransplantation hyperacute rejection study. The study determinedthat Human IgG caused a dose-dependent decrease in deposition ofcomplement protein iC3b and a decrease in formation of C3 convertase.Furthermore, the infusion of IgG was found to prevent hyperacuterejection of porcine hearts transplanted into the primates. Magee J. C.et al., J.Clin.Invest 1995 96(5):2404-2412. U.S. Pat. No. 5,851,528, Koet al., issued Dec. 22, 1998, U.S. Pat. No. 5,679,546, Ko et al., issuedOct. 21, 1997, and U.S. Pat. No. 5,627,264, Fodor et al., issued May 6,1997, describe chimeric proteins useful in inhibiting complementactivation and describe methods to treat adverse conditions related tocomplement mediated inflammation. Sims, et al., U.S. Pat. No. 5,550,508,issued Aug. 27, 1996, describes polypeptides which act to inhibitcomplement C5b-9 complex activity. The protein is an 18 kDA proteinfound on the surface of human erythrocytes and is described as beinguseful in treating immune disease states when administered in effectiveamounts.

Magee, J. C. et al., J. Clin. Invest. 1995 96(5):2404-12, investigatedthe use of immunoglobulin to prevent complement-mediated hyperacuterejection in swine-to-primate xenotransplantation. In the study humanIgG was added to human serum and was found to cause a dose-dependentdecrease in the deposition of iC3b, cytotoxicity, and heparin sulfaterelease when the serum was incubated with porcine endothelial cells. Itappears as if the decrease was caused by a decrease in the formation ofC3 convertase on the endothelial cells. Furthermore, infusion ofpurified human IgG into primates prevented hyperacute rejection ofporcine hearts in a xenotransplantation. Magee et al., determined thatsuch results support the use of IgG as a therapeutic agent inhumoral-mediated disease conditions.

U.S. Pat. No. 4,374,831, Joseph et al., issued Feb. 22, 1983, U.S. Pat.No. 4,087,548, Lenhard et al., issued May 2, 1978, U.S. Pat. No.4,021,545, Nair et al., issued May 3, 1977, and U.S. Pat. No. 3,998,957,Conrow et al., issued Dec. 21, 1976, all describe chemical moleculeswhich are useful as inhibitors and/or modulators of complement. Josepheet al., describe Bis-(β-D-glucopyranosyl-1-oxy)-arylene sulfatederivatives and methods for modulating complement in a warm bloodedanimal using pharmaceutical compounds comprising such molecules. Lenhardet al., describe complement inhibitory compounds such as C-substitutedtrisulfonic acids, acid ureides, and oxalyl amides and methods forinhibiting the complement system in a warm blooded animal byadministering complement inhibitory amounts of the compounds comprisingsuch molecules. Nair et al., claim methods for inhibiting the complementsystem in a warm blooded animal by using compositions comprising Inulinpoly(H-sulfate). Conrow et al., describe1,1′-[ureylenebis(sulfo-p-phenylene)]bis{sulfo-1H,8H-indazolo{2,3,4-cde]benzotriazol-9-iumhydroxide}, bis(inner salts), and tetra salts as useful complementinhibitors.

In WO 95/32719 (1995), Galbraith describes the use of phosphorothioateoligonucleotides for depleting complement. The approach describedinvolves administering, to a primate, an oligonucleotide 2 to 50nucleotides in length containing at least one phosphorothioateinternucleotide linkage, thereby stimulating vasodilation, and reducingcomplement activity by depleting complement.

Lin et al., WO 97/42317, describe oligonucleotides (aptamers) havingphosphorothioate and/or substituted phosphonate linkages that are 37-61base pairs in length which bind complement protein C3b and may be useddiagnostically in vivo or in vitro to detect C3b in a biological sample.The aptamers may be used therapeutically to inhibit undesirableC3b-mediated complement events such as inflammation.

There has been and continues to be a long-felt need for methods for theinhibition and/or modulation of the complement mediated immune responseusing modified oligonucleotide compounds that might incorporatemodifications for improving characteristics such as compound stabilityand cellular uptake. Such methods would be useful for therapeuticallyand prophylactically, as well as for diagnostic reagents and researchreagents including reagents for the study of both cellular and in vitroevents.

SUMMARY OF THE INVENTION

This invention relates to methods for modulating complement activation.These methods incorporate using modified oligonucleotides capable ofinhibiting complement activation and/or initiating complementactivation, depending on oligonucleotide concentration, and therebyprovide a method for modulating complement. The methods are also usefultherapeutically for the treatment of abnormal and/or undesirableconditions which can arise as a result of complement activation. Otheruses for the methods presently described, such as for example asdiagnostics and research reagents, are also included.

Thus in a first aspect, the present invention features methods formodulating complement activation by independently administering totissue, cells, cell/tissue culture, a bodily fluid, or a biologicalsample, a modified oligonucleotide in two different concentrations.Preferably, the first administered concentration initiates complementactivation and the second concentration inhibits complement activation,although it is within the scope of the invention to have the firstadministered concentration inhibit complement activation and the secondadministered concentration initiate complement activation. In anembodiment of the invention, the initiating concentration of modifiedoligonucleotide is no greater than 80 μg/ml, and more preferably between50 μg/ml and 80 μg/ml, and the inhibitory concentration of modifiedoligonucleotide is at least 200 μg/ml, and more preferably between 250μg/ml and 300 μg/ml.

Most preferably the inhibitory concentration is greater than theactivating concentration. Within the scope of the invention are furtherconcentrations determined through methods such as titration whereinconcentration levels are determined based on the condition or extent towhich complement modulation is desired.

In preferred embodiments the methods are performed in vitro or ex vivoand are preferably performed on a bodily fluid sample or a biologicalsample such as for example a mammalian blood or serum sample. Morepreferably the mammalian blood or serum sample is from a primate, andmost preferably the sample is from a human. Within this embodiment thebiological fluid sample includes samples of tissue or cells, wherein thesample also contains complement components.

In other embodiments the methods are performed in vivo in a mammal.Preferably the mammal is a primate and most preferably the primate is ahuman.

In an additional embodiment the present invention provides methods fortreating a human subject determined to have an abnormal or undesirablecondition associated with complement activation by administering a firstand second concentration of an oligonucleotide compound which modulatescomplement activity. Preferably the compound is administered in a firstinitiating concentration and a second inhibitory concentration. Theoligonucleotide preferably contains one or more phosphorothioatemodifications. It is preferred that the modulating concentrations aresimilar to those discussed above for both the first and secondadministration.

In more preferred embodiments the methods for treating are performed exvivo on a cell culture, tissue sample, bodily fluid or a biologicalsample taken from a human. Most preferably the methods are performed invivo in a human subject having an abnormal or undesirable conditionassociated with complement activation as determined by a licensedphysician.

In an additional aspect the methods described herein feature anoligonucleotide which contains at least one phosphorothioate (P═S)modification and which modulates complement activity by initiatingcomplement activation at a first oligonucleotide concentration andinhibiting complement activation at a second oligonucleotideconcentration. The inhibition and initiation concentrations of themodified oligonucleotide are independent and separate measurements andare not considered to be the total concentration of oligonucleotide in asample or host. What is most preferred is that the first (initiating)concentration of the modified oligonucleotide be lower than the second(inhibiting) concentration of the same oligonucleotide.

It is preferred that the concentration of oligonucleotide required toinitiate complement activation be less than or equal to 80 μg/ml, andmore preferably the concentration required to initiate complementactivation is between 50 μg/ml and 80 μg/ml. Within this same aspect ofthe invention, it is presently preferred that the concentration ofoligonucleotide required to inhibit complement activation be at least200 μg/ml; more preferably the concentration required to inhibitcomplement activation is between 250 μg/ml and 300 μg/ml. In regard tothe first and second concentrations of oligonucleotide the preferredembodiments are not considered limiting.

Included in the invention, are methods for modulating complementactivation in a cell culture, tissue or a bodily fluid by administeringa modified oligonucleotide compound which inhibits complement activationand which contains at least one phosphorothioate modification and isconjugated to a complement activation inhibitory molecule. In preferredembodiments the methods are performed in vitro or ex vivo and arepreferably performed on a bodily fluid sample, cell culture or abiological sample such as for example a mammalian blood or serum sample.More preferably the mammalian blood or serum sample is from a primate,and most preferably the sample is from a human. Within this embodimentthe biological fluid sample includes samples of tissue or cells, whereinthe sample also contains complement components.

Preferably, the modified oligonucleotide contains at least onephosphorothioate modification and is conjugated to a complementactivation inhibitory molecule. More preferably the complementactivation inhibitory molecule is a serum, vascular or cellular ligand,small complement binding molecule, or a complement specific ligand. Mostpreferably the complement activation inhibitory molecule bindscomplement Factor H. Preferably, the modified oligonucleotide to whichthe complement activation inhibitory molecule is bound is up to 60oligonucleotides in length; in more preferred embodiments the modifiedoligonucleotide which inhibits complement activation is between 8 and 30nucleotides in length. In an additional aspect, the invention features amodified oligonucleotide compound which inhibits complement activation.

In other embodiments the methods are performed in vivo in a mammal.Preferably the mammal is a primate and most preferably the primate is ahuman.

The term “independently administering” as used herein means providingone concentration (inhibitory or initiating) of modified oligonucleotideto the host and/or host cells at a time in order to modulate complementactivity. The manner in which the modified oligonucleotide isadministered may be selected from, but is not limited to: intravenousinfusion, needle injection, topical, needle-free injection as in, forexample, an injection using a device like the Medi-Jector™, and byaliquots using a pipette or similar device.

By use of term “culture” is meant the propagation of cells. Variousculture methods exist and are included within the scope of theinvention, methods such as, but not limited to, tissue culture methods,batch culture methods, enrichment culture methods, and ex vivo culturemethods. In all culture methods the cells to be propagated should be ina nutritive environment which allows for continued cell growth,complement activation and/or inactivation. Tissue and cell culturemethods are well understood in the art as these methods have beenregularly practiced in various scientific fields for years. Suchcultures may be propagated in natural serum or in artificial serum asdescribed for example in U.S. Pat. No. 4,657,866 to Kumar, Sudhir.Inasmuch as a culture represents a group of cells being observed foreffects relating to complement activation or inhibition, included withinthe scope of the invention are cultures of cells on or in a host, suchas a mass of burned tissue or cells, or a tumor growth, which mustremain on or in the host to be propagated.

By the phrase “monitoring complement activity” is meant measuringproducts of the proteolytic complement cascade. Such products to bemeasured include, but are not limited to, complement proteins: C5a, C3a,and C4a. Methods for measuring products of the complement cascade aredisclosed hereinbelow and can include antibody specific labeling ofcomplement proteins C5A, C3a and C4a and performing ELISA assays todetermine the relative concentration of the split products formed. Ingeneral monitoring of complement activity is performed on a biologicalsample that has been taken from a subject, patient or host, such as forexample a serum or blood sample or other bodily fluid.

Further aspects of the invention are described within the description ofthe preferred embodiments. The summary of the invention described aboveis not limiting and other features and advantages of the invention willbe apparent from the following detailed description of the invention andfrom the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings will herein briefly be described.

FIG. 1 is a line graph which shows the activation of complement inmonkey serum by the addition of 100 μg/ml of a phosphorothioateoligonucleotide (ISIS 2302; SEQ ID NO:1) of the invention as measured bythe amounts of complement components compared to baseline levels.

FIG. 2 is a line graph which shows the measurement of complementcomponent C3a to determine complement activation over a range of addedconcentrations of a phosphorothioate oligonucleotide of the presentinvention (ISIS 2302; SEQ ID NO:1)

FIG. 3 is a line graph which shows the inhibition of complementactivation as measured by the amount of complement component C3a inmonkey serum after stimulating complement activation with cobra venomfactor or zymosan. Inhibition is measured over a concentration range ofadded phosphorothioate oligonucleotide (ISIS 2302; SEQ ID NO:1) asdescribed herein.

FIG. 4 is a line graph which shows the inhibition of complementactivation as measured by the amount of complement component C3a inhuman serum after stimulating complement activation with cobra venomfactor or zymosan. Inhibition is measured over a concentration range ofadded phosphorothioate oligonucleotide (ISIS 2302; SEQ ID NO:1) asdescribed herein.

FIG. 5 shows a schematic representation of the complement system.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Complement comprises a proteolytic cascade of serum and membraneproteins which are part of the immune system and are responsible forprotecting hosts against foreign pathogens.

The complement system has powerful cytolytic activity which can damagean individual's own cells and should therefore be a target formodulation in order to reduce injury in various autoimmune events. Inmost cases individuals possess proteins which can control the extent ofcomplement activation in serum or on the surfaces of “self” cells. Mostof the proteins which inhibit complement activation in serum serve tolimit the generation of complement fragments such as C4b and C3b.Proteins such as C1-inhibitor, C4-binding protein, Factor H, and FactorI serves as normal inhibitors of complement in “normal” individuals. Inan non-limiting example, C1 inhibitor is not present due to a geneticdeletion or point mutations that produce an inactive form and results inhereditary angioedema of which there is also an acquired form usuallydue to auto-antibody to C1 INH. In such an abnormal condition such asangioedema as in others there is a need to help modulate complement inorder to reduce the damage that can occur.

As described above, various methods have been investigated to inhibitcomplement activation. Wuillemin et al., J. Immunol 15;159(4):1953-60,(1997), describes the use of glycosaminoglycans, such as heparin, toinhibit the interaction of complement component C1q with othercomplement activators and the assembly of the classical and alternativepathway C3 convertases. Naturally occurring glycosaminoglycans such asdextran sulfates, heparin, N-acetylheparin, heparan sulfate, dermatansulfate, and chondroitin sulfates A and C were studied to determinetheir effectiveness at inhibiting the deposition of C4 and C3 onimmobilized aggregated human IgG and to reduce fluid phase formation ofC4b/c and C3b/c. In the study it was concluded that glycosaminoglycanssuch as the low molecular weight dextran sulfate (m.w. 5000) may serveas candidates for pharmacological manipulation of complement activationvia potentiation of C1 inhibitor.

We have found that activation of the alternative pathway of complementoccurs following the intravenous infusion of modifiedoligodeoxynucleotides (P═S oligo). By using monkey serum and whole bloodwe determined that modified oligonucleotides cause an increase incomplement products Bb, C3a, and C5a. The concentration that was foundto activate complement in these experimental systems was found to be upto roughly 50 μg/ml. By using the same modified oligonucleotide (ISIS2302; SEQ ID NO:1) it was determined that at concentrations of roughly250 μg/ml and greater complement activation was inhibited in both theclassical and alternative pathways as indicated by a reduction incomplement components Bb, C3a, and C5a.

The present invention provides methods for modulating complementactivity using modified oligonucleotides. The invention provides methodsfor using modified oligonucleotides which involve administering theoligonucleotides at one concentration to initiate complement activationand at another concentration to inhibit complement activation. Theoligonucleotides of the present invention are modified to have improvedpharmacokinetic properties. The methods described herein are useful astherapeutics for the treatment, prevention or diagnosis of abnormaland/or undesirable conditions which can arise as a result of complementmediated inflammatory effects.

By “abnormal and/or undesirable conditions” is meant any conditions thathave an inflammatory, immune or autoimmune component associated with theactivation of the complement cascade. An abnormal and/or undesirablecondition can be, but is not limited to: myasthenia gravis, immunecomplex excess syndromes such as systemic lupus, erythematosus,ischemia-reperfusion states, angioedema, hyper-acute rejection oftransplants, organ failure conditions such as adult respiratory distresssyndrome, Alzheimer's disease and related neurodegenerative disorders.Such conditions are generally determined by registered physicians.

Other envisioned treatments are for conditions in which a host isinvaded by a foreign body which avoids the complement system and whichmay be targeted by an oligonucleotide according to the present inventionin order to activate the complement system and eliminate the invadingmolecule.

Modifications of Oligonucleotides

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of ribonucleic acid or deoxyribonucleic acid.This term includes oligonucleotides composed of naturally-occurringnucleobases, sugars and covalent intersugar (backbone) linkages as wellas oligonucleotides having non-naturally-occurring portions whichfunction similarly. Such “modified” or substituted oligonucleotides areoften preferred over native forms because of desirable properties suchas, for example, enhanced cellular uptake, enhanced binding to target,increased stability in the presence of nucleases and an increase inbioavailability. In the present invention, oligonucleotides having atleast one phosporothioate modification are preferred.

Within the concepts of “oligonucleotides” and “modified”oligonucleotides, the present invention also includes compositionsemploying oligonucleotide compounds which are chimeric compounds.“Chimeric” oligonucleotide compounds or “chimeras,” in the context ofthis invention, are nucleic acid compounds, particularlyoligonucleotides, which contain two or more chemically distinct regions,each made up of at least one monomer unit, i.e., a nucleotide in thecase of an oligonucleotide compound. These oligonucleotides typicallycontain at least one region wherein the oligonucleotide is modified soas to confer upon the oligonucleotide increased resistance to nucleasedegradation, increased cellular uptake, and/or consist of an oligomericsequence known to modify complement activation. An additional region ofthe oligonucleotide may serve as a substrate for enzymes capable ofcleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is acellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex.Activation of RNase H, therefore, results in cleavage of the RNA target,thereby greatly enhancing the efficiency of oligonucleotide inhibitionof gene expression. Consequently, comparable results can often beobtained with shorter oligonucleotides when chimeric oligonucleotidesare used, compared to phosphorothioate oligodeoxynucleotides hybridizingto the same target region. Cleavage of the RNA target can be routinelydetected by gel electrophoresis and, if necessary, associated nucleicacid hybridization techniques known in the art. RNase H-mediated targetcleavage is distinct from the use of ribozymes to cleave nucleic acids.

By way of example, such “chimeras” may be “gapmers,” i.e.,oligonucleotides in which a central portion (the “gap”) of theoligonucleotide serves as a substrate for, e.g., RNase H, and the 5′ and3′ portions (the “wings”) are modified in such a fashion so as to havegreater affinity for, or stability when duplexed with, the target RNAmolecule but are unable to support nuclease activity (e.g., 2′-fluoro-or 2′-methoxyethoxy-substituted). Other chimeras include “hemimers,”that is, oligonucleotides in which the 5′ portion of the oligonucleotideserves as a substrate for, e.g., RNase H, whereas the 3′ portion ismodified in such a fashion so as to have greater affinity for, orstability when duplexed with, the target RNA molecule but is unable tosupport nuclease activity (e.g., 2′-fluoro- or 2′-methoxyethoxy-substituted), or vice-versa.

A number of chemical modifications to oligonucleotides that confergreater oligonucleotide:RNA duplex stability have been described byFreier et al. (Nucl. Acids Res., 1997, 25, 4429). Such modifications arepreferred for the RNase H-refractory portions of chimericoligonucleotides and may generally be used to enhance the affinity of anantisense compound for a target RNA. In this way, in a preferredembodiment, a chimeric molecule comprised of a modified oligonucleotidewhich modulates complement and an antisense portion may be administeredin order to target a specific RNA molecule and modulate complementmediated adverse effects.

Chimeric modified oligonucleotide compounds of the invention may beformed as composite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleosides and/or oligonucleotide mimetics asdescribed above, ligand-oligonucleotide constructs, or complementprotein-oligonucleotide constructs as described herein. Some of thesecompounds have also been referred to in the art as hybrids or gapmers.Representative United States patents that teach the preparation of someof these hybrid structures include, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and U.S. Pat. No.5,700,922, certain of which are commonly owned, and each of which isherein incorporated by reference, and commonly owned and allowed U.S.patent application Ser. No. 08/465,880, filed on Jun. 6, 1995, now U.S.Pat. No. 5,955,589 also herein incorporated by reference.

Modifications to an oligonucleotide molecule can alter the concentrationof the molecule required to elicit the effect for which the molecule isdesigned. Non limiting examples include varying the amount ofphosphorothioate linkages in the oligonucleotide or altering theoligonucleotide base composition and chemistry such as in thepreparation of CpG oligodeoxynucleotides as described by Krieg et al.,Nature 1995 374:546-549, Weiner et al., Proc. Natl. Acad. Sci. USA 199794:10833-10837, Liu, HM et al., Blood 1998 15;92(10):3730-3736, Boggs,RT et al., Antisense Nucleic Acid Drug Dev 1997 7(5):461-471, and Klineet al., J.Immunol 1998 15;160(6):2555-2559, which are all herebyincorporated herein in their entirety including any figures anddrawings.

The present invention also includes compositions employingoligonucleotides that are substantially chirally pure with regard toparticular positions within the oligonucleotides. Examples ofsubstantially chirally pure oligonucleotides include, but are notlimited to, those having phosphorothioate linkages that are at least 75%Sp or Rp (Cook et al., U.S. Pat. No. 5,587,361) and those havingsubstantially chirally pure (Sp or Rp) alkylphosphonate, phosphoramidateor phosphotriester linkages (Cook, U.S. Pat. Nos. 5,212,295 and5,521,302).

Oligonucleotides may contain modifications of the backbone sugar and/ornucleobase, singly or in combination. Specific examples of somepreferred backbone modified oligonucleotides envisioned for thisinvention include those containing phosphorothioates (P═Soligonucleotides), phosphotriesters, methyl phosphonates, short chainalkyl or cycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. Oligonucleotides having modifiedbackbones include those that retain a phosphorus atom in the backboneand those that do not have a phosphorus atom in the backbone. For thepurposes of this specification, and as sometimes referenced in the art,modified oligonucleotides that do not have a phosphorus atom in theirinternucleoside backbone can also be considered to be oligonucleosides.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andborano-phosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050, certain of which are commonly owned with thisapplication, and each of which is herein incorporated by reference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, certain of which are commonly ownedwith this application, and each of which is herein incorporated byreference.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

Other preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S—or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyland alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. A preferred modificationincludes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as2′-O—(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995,78, 486-504) i.e., an alkoxyalkoxy group. A further preferredmodification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE, as described in examples hereinbelow, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples hereinbelow.

Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugar structures include, but are not limited to, U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, certain of which are commonly ownedwith the instant application, and each of which is herein incorporatedby reference in its entirety.

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substitutedadenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B. , ed., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyl-adenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds., Antisense Research andApplications, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; and 5,681,941, certain of which are commonly ownedwith the instant application, and each of which is herein incorporatedby reference, and U.S. Pat. No. 5,750,692, which is commonly owned withthe instant application and also herein incorporated by reference.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. Such moieties include but are not limitedto lipid moieties such as a cholesterol moiety (Letsinger et al., Proc.Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan etal., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660,306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770),a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20,533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues(Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al.,FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75,49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide.

Another preferred additional or alternative modification of theoligonucleotides of the invention involves chemically linking to theoligonucleotide one or more lipophilic moieties which enhance thecellular uptake of the oligonucleotide. Such lipophilic moieties may belinked to an oligonucleotide at several different positions on theoligonucleotide. Some preferred positions include the 3′ position of thesugar of the 3′ terminal nucleotide, the 5′ position of the sugar of the5′ terminal nucleotide, and the 2′ position of the sugar of anynucleotide. The N⁶ position of a purine nucleobase may also be utilizedto link a lipophilic moiety to an oligonucleotide of the invention(Gebeyehu, G., et al., Nucleic Acids Res., 1987, 15, 4513). Suchlipophilic moieties include but are not limited to a cholesteryl moiety(Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553), cholicacid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053), athioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad.Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993,3, 2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992,20, 533), an aliphatic chain, e.g., dodecandiol or undecyl residues(Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov et al., FEBSLett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49), aphospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990,18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277, 923). Oligonucleotidescomprising lipophilic moieties, and methods for preparing sucholigonucleotides, as disclosed in U.S. Pat. Nos. 5,138,045, 5,218,105and 5,459,255, the contents of which are hereby incorporated byreference in their entirety.

In other preferred embodiments the compound may be a ligand conjugatedoligomeric compound having improved pharmacokinetic properties. Sucholigomeric compounds are prepared having covalently attached ligands orproteins that bind reversibly to or interact with one or more serum,vascular or cellular proteins. This reversible binding is expected todecrease urinary excretion, increase serum half life and greatlyincrease the distribution of oligomeric compounds thus conjugated. Inthe case of binding a complement protein, such as for example complementFactor H or a ligand thereof, in the context of the present inventionthe binding is to further inhibit complement activity. The binding ofparticular drugs to plasma protein has been previously shown to enhancethe disposition and efficacy of drugs (Herve et al., Clin.Pharmacokinet., 1994, 26:44).

An oligomeric agent should be able to overcome inherent factors such asrapid degradation in serum, short half life in serum and rapidfiltration by the kidneys with subsequent excretion in the urine.Oligonucleotides that overcome these inherent factors have increasedserum half lives, distribution, cellular uptake and hence improvedefficacy. These enhanced pharmacokinetic parameters have been shown forselected drug molecules that bind plasma proteins (Olson and Christ,Annual Reports in Medicinal Chemistry, 1996, 31:327). Two proteins thathave been studied more than most are human serum albumin (HSA) andα-1-acid glycoprotein. HSA binds a variety of endogenous and exogenousligands with association constants typically in the range of 10⁴ to 10⁶M⁻¹. Association constants for ligands with α-1-acid glycoprotein aresimilar to those for HSA.

At least for therapeutic purposes, oligonucleotides should have a degreeof stability in serum to allow distribution and cellular uptake. Theprolonged maintenance of therapeutic levels of oligomeric agents inserum will have a significant effect on the distribution and cellularuptake and unlike conjugate groups that target specific cell receptorsthe increased serum stability will affect all cells. Numerous effortshave focused on increasing the cellular uptake of oligonucleotidesincluding increasing the membrane permeability via conjugates andcellular delivery of oligonucleotides.

Many drugs reversibly bind to plasma proteins. A representative list,which is not meant to be inclusive, includes: aspirin, warfarin,phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen,(S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoicacid, flufenamic acid, folinic acid, benzothiadiazides, chlorothiazide,diazepines (such as for example fludiazepam and diazepam) indomethacin,barbiturates (such as for example quinalbarbitone), cephalosporins,sulfa drugs, antidiabetics (such as for example tolbutamide),antibacterials (such as for example a group of quinolones; nalidixicacid and cinoxacin) and several antibiotics. Serum albumin is the mostimportant protein among all plasma proteins for drug binding, althoughbinding to other proteins (for example, macroglobulin G₂,immunoglobulins, lipoproteins, alpha-1-acid glycoprotein, thrombin) isalso important.

Ligands that bind serum, vascular or cellular proteins may be attachedvia an optional linking moiety to one or more sites on anoligonucleotide of the invention. These sites include one or more of,but are not limited to, the 2′ position, 3′-position, 5′-position, theinternucleotide linkage, and a nucleobase atom of any nucleotideresidue. The attachment of ligands to such structures can be performed,according to some preferred embodiments of the invention, using alinking group, or without the use of such a linking group. Preferredlinking groups of the invention include, but are not limited to,6-aminoalkoxy linkers, 6-aminoalkylamino linkers, cysteamine,heterobifunctional linkers, homobifunctional linkers, and a universallinker (derived from 3-dimethoxytrityloxy-2-aminopropanol). Aparticularly preferred linking group for the synthesis of ligandconjugated oligonucleotides of the invention is a 6-aminohexyloxy group.A variety of heterobifunctional and homobifunctional linking moietiesare available from Pierce Co. (Rockford, Ill.). Such heterobifunctionaland homobifunctional linking moieties are particularly useful inconjunction with the 6-aminoalkoxy and 6-aminoalkylamino moieties toform extended linkers useful for linking ligands to a nucleoside.Further useful linking groups that are commercially available are5′-Amino-Modifier C6 and 3′-Amino-Modifier reagents, both available fromGlen Research Corporation (Sterling, Va.). 5′-Amino-Modifier C6 is alsoavailable from ABI (Applied Biosystems Inc., Foster City, Calif.) asAminolink-2, while the 3′-Amino-Modifier is also available from ClontechLaboratories Inc. (Palo Alto, Calif.). In addition, a nucleotide analogbearing a linking group pre-attached to the nucleoside is commerciallyavailable from Glen Research Corporation under the tradename“Amino-Modifier-dT.” This nucleoside-linking group reagent, a uridinederivative having an [N(7-trifluoroacetylaminoheptyl)3-acrylamido]substituent group at the 5 position of the pyrimidine ring, issynthesized as per the procedure of Jablonski et al. (Nucleic AcidResearch, 1986, 14:6115). The present invention also includes asnucleoside analogs adenine nucleosides functionalized to include alinker on the N6 purine amino group, guanine nucleosides functionalizedto include a linker at the exocyclic N2 purine amino group, and cytosinenucleosides functionalized to include a linker on either the N4pyrimidine amino group or the 5 pyrimidine position. Such nucleosideanalogs are incorporated into oligonucleotides with a ligand attached tothe linker either pre- or post-oligomerization.

In a preferred embodiment of the present invention ligand molecules areselected for conjugation to oligonucleotides on the basis of theiraffinity for one or more complement proteins. These proteins may beserum, vascular or cellular proteins. Serum proteins are proteins thatare present in the fluid portion of the blood, obtained aftercoagulation and removal of the fibrin clot and blood cells, asdistinguished from the plasma in circulating blood. Vascular proteinsare proteins that are present in portions of the vascular systemrelating to or containing blood vessels. Cellular proteins are membraneproteins which have at least a portion of the protein extendingextracellularly and assisting in the process of endocytosis.

Many ligands having an affinity for proteins are well documented in theliterature and are amenable to use in the present invention. A preferredgroup of ligands are small molecules including drug moieties. Accordingto the present invention, drug moieties include, but are not limited to,warfarin and coumarins including substituted coumarins, isocoumarinderivatives, 7-anilinocoumarin-4-acetic acid, profens includingibuprofen, enantiomers of ibuprofen (r-ibuprofen and s,-ibuprofen),ibuprofen analogs, ketoprofen, carprofen, etodolac, suprofen,indoprofen, fenbufen, arylpropionic acids, arylalkanoic acids,2-aryl-2-fluoropropionic acids, glibenclamide, acetohexamide,arylalkanoic acids, tolbutamide, gliclazide, metformin, curcumin,digitoxin, digoxin, diazepam, benzothiadiazides, chlorothiazide,diazepines, benzodiazepines, naproxen, phenyl butazone, oxyphenbutazone,dansyl amide, dansylsarcosine, 2,3,5-triiodobenzoic acid, palmitic acid,aspirin, salicylates, substituted salicylates, penicillin, flurbiprofen,pirprofin, oxaprozin, flufenamic acid, deoxycholic acid, glycyrrhizin,azathioprine, butibufen, ibufenac, 5-fluoro-1-typtaphan,5-fluoro-salicylic, acidazapropanazone, mefenamic acid, indomethacin,flufenamic acid, bilirubin, ibuprofen, lysine complexes, diphenylhydantoin, valproic acid, tolmetin, barbiturates (such as, for example,quinalbarbitone), cephalosporins, sulfa drugs, antidiabetics (such as,for example, tolbutamide), antibacterials (such as, for example,quinolones, nalidixic acid and cinoxacin) and several antibiotics.

In one embodiment of the present invention the drug moiety bears acarboxylic acid group. In another embodiment of the present inventionthe drug moiety is a propionic acid derivative.

In one preferred embodiment of the invention the protein for binding aligand conjugated oligomeric compound is a serum protein. It ispreferred that the serum protein bound by a conjugated oligomericcompound is an immunoglobulin (an antibody). Preferred immunoglobulinsare immunoglobulin G and immunoglobulin M. Immunoglobulins are known toappear in blood serum and tissues of vertebrate animals. A morepreferred protein for binding to a ligand conjugated oligomer isalbumin.

In another embodiment of the invention the serum protein for binding bya conjugated oligomeric compound is a lipoprotein. Lipoproteins areblood proteins having molecular weights generally above 20,000 thatcarry lipids and are recognized by specific cell surface receptors. Theassociation with lipoproteins in the serum will initially increasepharmacokinetic parameters such as half life and distribution. Asecondary consideration is the ability of lipoproteins to enhancecellular uptake via receptor-mediated endocytosis.

In yet another embodiment the serum protein for binding by a ligandconjugated oligomeric compound is α-2-macroglobulin. In yet a furtherembodiment the serum protein targeted by a ligand conjugated oligomericcompound is α-1-glycoprotein.

As used herein, the term “protected” means that the indicated moiety hasa protecting group appended thereon. In some preferred embodiments ofthe invention compounds contain one or more protecting groups. A widevariety of protecting groups can be employed in the methods of theinvention. In general, protecting groups render chemical functionalitiesinert to specific reaction conditions, and can be appended to andremoved from such functionalities in a molecule without substantiallydamaging the remainder of the molecule.

Representative hydroxyl protecting groups, for example, are disclosed byBeaucage et al. (Tetrahedron, 1992, 48:2223-2311). Further hydroxylprotecting groups, as well as other representative protecting groups,are disclosed in Greene and Wuts, Protective Groups in OrganicSynthesis, Chapter 2, 2d ed., John Wiley & Sons, New York, 1991, andOligonucleotides And Analogues A Practical Approach, Ekstein, F. Ed.,IRL Press, N.Y., 1991, each of which is hereby incorporated by referencein its entirety.

Examples of hydroxyl protecting groups include, but are not limited to,t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl,p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2, 6-dichlorobenzyl,diphenylmethyl, p,p′-dinitrobenzhydryl, p-nitrobenzyl, triphenylmethyl,trimethylsilyl, triethylsilyl, t-butyldimethylsilyl,t-butyldiphenylsilyl, triphenylsilyl, benzoylformate, acetate,chloroacetate, trichloroacetate, trifluoroacetate, pivaloate, benzoate,p-phenylbenzoate, 9-fluorenylmethyl carbonate, mesylate and tosylate.

Amino-protecting groups stable to acid treatment are selectively removedwith base treatment, and are used to make reactive amino groupsselectively available for substitution. Examples of such groups are theFmoc (E. Atherton and R. C. Sheppard in The Peptides, S. Udenfriend, J.Meienhofer, Eds., Academic Press, Orlando, 1987, volume 9, p.1) andvarious substituted sulfonylethyl carbamates exemplified by the Nscgroup (Samukov et al., Tetrahedron Lett, 1994, 35:7821; Verhart andTesser, Rec. Trav. Chim. Pays-Bas, 1987, 107:621).

Additional amino-protecting groups include, but are not limited to,carbamate-protecting groups, such as 2-trimethylsilylethoxycarbonyl(Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl(BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc),and benzyloxycarbonyl (Cbz); amide-protecting groups, such as formyl,acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl;sulfonamide-protecting groups, such as 2-nitrobenzenesulfonyl; andimine- and cyclic imide-protecting groups, such as phthalimido anddithiasuccinoyl. Equivalents of these aminoprotecting groups are alsoencompassed by the compounds and methods of the present invention.

In a preferred embodiment of the present invention oligonucleotides areprovided including a number of linked nucleosides wherein at least oneof the nucleosides is a 2′-functionalized nucleoside having a ligandmolecule linked to the 2′-position of the nucleoside; a heterocyclicbase functionalized nucleoside having a ligand molecule linked to theheterocyclic base of the nucleoside, a 5′ terminal nucleoside having aligand molecule linked to the 5′-position of the nucleoside, a 3′terminal nucleoside having a ligand molecule linked to the 3′-positionof the nucleoside, or an inter-strand nucleoside having a ligandmolecule linked to an inter-stand linkage linking said inter-strandnucleoside to an adjacent nucleoside.

Ligand conjugated oligonucleotides may be synthesized by the use of anoligonucleotide that bears a pendant reactive functionality such as thatderived from the attachment of a linking molecule onto theoligonucleotide. This reactive oligonucleotide may be reacted directlywith commercially available ligands, ligands that are synthesizedbearing a variety of protecting groups, or ligands that have a linkingmoiety attached thereto. The methods of the present invention facilitatethe synthesis of ligand conjugated oligonucleotides by the use of, insome preferred embodiments, nucleoside monomers that have beenappropriately conjugated with ligands and that may further be attachedto a solid support material. Such ligand-nucleoside conjugatesoptionally attached to a solid support material are prepared accordingto some preferred embodiments of the methods of the present inventionvia reaction of a selected serum binding ligand with a linking moietylocated on a 2′, 3′, or 5′ position of a nucleoside or oligonucleotide.

The above described conjugation of ligands to oligomeric compounds hasbeen shown to increase the concentration of such compounds in serum.According to such methods, a drug moiety that is known to bind to aserum protein is selected and conjugated to an oligonucleotide, thusforming a conjugated oligonucleotide. This conjugated oligonucleotide isthen added to the serum.

Conjugation of a ligand also provides a way to increase the capacity ofserum for an oligonucleotide. According to such methods, a drug moietythat is known to bind to a serum protein is selected and conjugated toan oligonucleotide, thus forming a conjugated oligonucleotide. Thisconjugated oligonucleotide is then added to the serum.

Ligand conjugation can also increase the binding of an oligonucleotideto a portion of the vascular system. According to such methods, a drugmoiety that is known to bind to a vascular protein is selected. Thevascular protein selected is a protein which resides, in part, in thecirculating serum and, in part, in the non-circulating portion of thevascular system. This drug moiety is conjugated to an oligonucleotide toform a conjugated oligonucleotide, which is then added to the vascularsystem.

The oligonucleotides used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including Applied Biosystems. Any other means for such synthesismay also be employed; the actual synthesis of the oligonucleotides iswell within the talents of the routineer. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and 2′-alkoxy or 2′-alkoxyalkoxy derivatives,including 2′-O-methoxyethyl oligonucleotides (Martin, P., Helv. Chim.Acta, 1995, 78, 486-504). It is also well known to use similartechniques and commercially available modified amidites andcontrolled-pore glass (CPG) products such as biotin, fluorescein,acridine or psoralen-modified amidites and/or CPG (available from GlenResearch, Sterling Va.) to synthesize fluorescently labeled,biotinylated or other conjugated oligonucleotides.

Complement Modulation

The modified oligonucleotide compounds of the present invention includebioequivalent compounds, including pharmaceutically acceptable salts andprodrugs. This is intended to encompass any pharmaceutically acceptablesalts, esters, or salts of such esters, or any other compound which,upon administration to an animal including a human, is capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, for example, the disclosure is also drawnto pharmaceutically acceptable salts of the nucleic acids of theinvention and prodrugs of such nucleic acids.

“Pharmaceutically acceptable salts” are physiologically andpharmaceutically acceptable salts of the nucleic acids of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto (see,for example, Berge et al., “Pharmaceutical Salts,” J. of Pharma Sci.,1977, 66:1, which is incorporated herein by reference in its entirety).

For oligonucleotides, examples of pharmaceutically acceptable saltsinclude but are not limited to (a) salts formed with cations such assodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

The oligonucleotides of the invention may additionally or alternativelybe prepared to be delivered in a “prodrug” form. The term “prodrug”indicates a therapeutic agent that is prepared in an inactive form thatis converted to an active form (i.e., drug) within the body or cellsthereof by the action of endogenous enzymes or other chemicals and/orconditions. In particular, prodrug versions of the oligonucleotides ofthe invention are prepared as SATE ((S-acetyl-2-thioethyl) phosphate)derivatives according to the methods disclosed in WO 93/24510 toGosselin et al., published Dec. 9, 1993, which is incorporated herein byreference in its entirety.

For therapeutic or prophylactic treatment, oligonucleotides areadministered in accordance with this invention. Oligonucleotidecompounds of the invention may be formulated in a pharmaceuticalcomposition, which may include pharmaceutically acceptable carriers,thickeners, diluents, buffers, preservatives, surface active agents,neutral or cationic lipids, lipid complexes, liposomes, penetrationenhancers, carrier compounds and other pharmaceutically acceptablecarriers or excipients and the like in addition to the oligonucleotide.Such compositions and formulations are comprehended by the presentinvention.

Pharmaceutical compositions comprising the oligonucleotides of thepresent invention may include penetration enhancers in order to enhancethe alimentary delivery of the oligonucleotides. Penetration enhancersmay be classified as belonging to one of five broad categories, i.e.,fatty acids, bile salts, chelating agents, surfactants andnon-surfactants (Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, 8:91-192; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7:1). One or more penetrationenhancers from one or more of these broad categories may be included.

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional compatible pharmaceutically-activematerials such as, e.g., antipruritics, astringents, local anestheticsor anti-inflammatory agents, or may contain additional materials usefulin physically formulating various dosage forms of the composition ofpresent invention, such as dyes, flavoring agents, preservatives,antioxidants, opacifiers, thickening agents and stabilizers. However,such materials, when added, should not unduly interfere with thebiological activities of the components of the compositions of theinvention.

Regardless of the method by which the oligonucleotides of the inventionare introduced into a patient, colloidal dispersion systems may be usedas delivery vehicles to enhance the in vivo stability of theoligonucleotides and/or to target the oligonucleotides to a particularorgan, tissue or cell type. Colloidal dispersion systems include, butare not limited to, macromolecule complexes, nanocapsules, microspheres,beads and lipid-based systems including oil-in-water emulsions,micelles, mixed micelles, liposomes and lipid:oligonucleotide complexesof uncharacterized structure. A preferred colloidal dispersion system isa plurality of liposomes. Liposomes are microscopic spheres having anaqueous core surrounded by one or more outer layers made up of lipidsarranged in a bilayer configuration (see, generally, Chonn et al.,Current Op. Biotech., 1995, 6, 698). Liposomal modified oligonucleotidecompositions are prepared according to the disclosure of co-pending U.S.patent application Ser. No. 08/961,469 to Hardee et al., filed Oct. 31,1997, U.S. Pat. No. 6,083,923, incorporated herein by reference in itsentirety.

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic, vaginal, rectal,intranasal, epidermal and transdermal), oral or parenteral, or byaliquots using a pipette or the like. Parenteral administration includesintravenous drip, injection or infusion, subcutaneous, intraperitonealor intramuscular injection, pulmonary administration, e.g., byinhalation or insufflation, or intracranial, e.g., intrathecal orintraventricular, administration. Injection includes both needleinjection and needle-free injection as in, for example, an injectionusing a device like the Medi-Jector™. For oral administration, it hasbeen found that oligonucleotides with at least one 2′-substitutedribonucleotide are particularly useful because of their absorption anddistribution characteristics. U.S. Pat. No. 5,591,721 issued to Agrawalet al. Oligonucleotides with at least one 2′-O-methoxyethyl modificationare believed to be particularly useful for oral administration.

Formulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.Coated condoms, gloves and the like may also be useful.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable.

Compositions for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives.

The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 μg to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 μg to 100 g per kgof body weight, once or more daily, to once every 20 years.

By “ex vivo” is meant removing a sample of blood, serum and/or bonemarrow from a subject in need of complement modulation, treating thesample with the modified oligonucleotide described herein, and returningthe sample to the subject.

Thus, in the context of this invention, by “therapeutically effectiveamount” is meant the amount of the compound which is required to have atherapeutic effect on the treated mammal. This amount, which will beapparent to the skilled artisan, will depend upon the type of mammal,the age and weight of the mammal, the type of disease to be treated,perhaps even the gender of the mammal, and other factors which areroutinely taken into consideration when treating a mammal with adisease. A therapeutic effect is assessed in the mammal by measuring theeffect of the compound on the disease state in the animal. For example,if the disease to be treated is an ischaemia-reperfusion event, areduction in tissue damage is an indication that the administered dosehas a therapeutic effect. In an example of a chimeric oligonucleotideusage, if the disease to be treated is psoriasis, a reduction orablation of the skin plaque and a reduced activation of complementoccurs this would also be an indication that the administered dose has atherapeutic effect. Similarly, in mammals being treated for cancer,therapeutic effects are assessed by measuring both the amount ofcomplement activation and the rate of growth or the size of the tumor,or by measuring the production of compounds such as cytokines,production of which is an indication of the progress or regression ofthe tumor.

The following examples illustrate the present invention and are notintended to limit the same.

EXAMPLES Example 1 Nucleoside Phosphoramidites for OligonucleotideSynthesis

Deoxy and 2′-alkoxy Amidites

2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropyl phosphoramidites arepurchased from commercial sources (e.g. Chemgenes, Needham Mass. or GlenResearch, Inc. Sterling Va.).

Other 2′-O-alkoxy substituted nucleoside amidites are prepared asdescribed in U.S. Pat. No. 5,506,351, herein incorporated by reference.For oligonucleotides synthesized using 2′-alkoxy amidites, the standardcycle for unmodified oligonucleotides is utilized, except the wait stepafter pulse delivery of tetrazole and base is increased to 360 seconds.

Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-C)nucleotides are synthesized according to published methods [Sanghvi, et.al., Nucleic Acids Research, 1993, 21, 3197-3203] using commerciallyavailable phosphoramidites (Glen Research, Sterling Va. or ChemGenes,Needham Mass.).

2′-Fluoro Amidites

2′-Fluorodeoxyadenosine Amidites

2′-fluoro oligonucleotides may be synthesized as described previously[Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] and U.S. Pat. No.5,670,633, herein incorporated by reference. Briefly, the protectednucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine is synthesizedutilizing commercially available 9-beta-D-arabin ofuranosyladenine asstarting material and by modifying literature procedures whereby the2′-alpha-fluoro atom is introduced by a S_(N)2-displacement of a2′-beta-trityl group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladenineis selectively protected in moderate yield as the3′,5′-ditetrahydropyranyl (THP) intermediate. Deprotection of the THPand N6-benzoyl groups is accomplished using standard methodologies andstandard methods may be used to obtain the 5′-dimethoxytrityl-(DMT) and5′-DMT-3′-phosphoramidite intermediates.

2′-Fluorodeoxyguanosine

The synthesis of 2′-deoxy-2′-fluoroguanosine is accomplished usingtetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate diisobutyryl-arabinofuranosylguanosine. Deprotection ofthe TPDS group is followed by protection of the hydroxyl group with THPto give diisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation is followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies may be used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

2′-Fluorouridine

Synthesis of 2′-deoxy-2′-fluorouridine is accomplished by themodification of a literature procedure in which2,2′-anhydro-1-beta-D-arabinofuranosyluracil is treated with 70%hydrogen fluoride-pyridine. Standard procedures may be used to obtainthe 5′-DMT and 5′-DMT-3′ phosphoramidites.

2′-Fluorodeoxycytidine

2′-deoxy-2′-fluorocytidine is synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures may be usedto obtain the 5′-DMT and 5′-DMT3′ phosphoramidites.

2′-O-(2-Methoxyethyl) Modified Amidites

2′-O-Methoxyethyl-substituted nucleoside amidites are prepared asfollows, or alternatively, as per the methods of Martin, P., HelveticaChimica Acta, 1995, 78, 486-504.

2,2′-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]

5-Methyluridine (ribosylthymine, commercially available through Yamasa,Choshi, Japan) (72.0 g, 0.279 M), diphenyl-carbonate (90.0 g, 0.420 M)and sodium bicarbonate (2.0 g, 0.024 M) are added to DMF (300 mL). Themixture is heated to reflux, with stirring, allowing the evolved carbondioxide gas to be released in a controlled manner. After 1 hour, theslightly darkened solution is concentrated under reduced pressure. Theresulting syrup is poured into diethylether (2.5 L), with stirring. Theproduct formed a gum. The ether is decanted and the residue is dissolvedin a minimum amount of methanol (ca. 400 mL). The solution is pouredinto fresh ether (2.5 L) to yield a stiff gum. The ether is decanted andthe gum is dried in a vacuum oven (60° C. at 1 mm Hg for 24 h) to give asolid that is crushed to a light tan powder (57 g, 85% crude yield). TheNMR spectrum is consistent with the structure, contaminated with phenolas its sodium salt (ca. 5%). The material is used as is for furtherreactions (or it can be purified further by column chromatography usinga gradient of methanol in ethyl acetate (10-25%) to give a white solid,mp 222-4° C.).

2′-O-Methoxyethyl-5-methyluridine

2,2′-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate(231 g, 0.98 M) and 2-methoxyethanol (1.2 L) may be added to a 2 Lstainless steel pressure vessel and placed in a pre-heated oil bath at160° C. After heating for 48 hours at 155-160° C., the vessel is openedand the solution evaporated to dryness and triturated with MeOH (200mL). The residue is suspended in hot acetone (1 L). The insoluble saltsmay be filtered, washed with acetone (150 mL) and the filtrateevaporated. The residue (280 g) is dissolved in CH₃CN (600 mL) andevaporated. A silica gel column (3 kg) is packed in CH₂Cl₂/acetone/MeOH(20:5:3) containing 0.5% Et₃NH. The residue is dissolved in CH₂Cl₂ (250mL) and adsorbed onto silica (150 g) prior to loading onto the column.The product is eluted with the packing solvent to give 160 g (63%) ofproduct. Additional material is obtained by reworking impure fractions.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) is co-evaporated withpyridine (250 mL) and the dried residue dissolved in pyridine (1.3 L). Afirst aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) is added andthe mixture stirred at room temperature for one hour. A second aliquotof dimethoxytrityl chloride (94.3 g, 0.278 M) is added and the reactionstirred for an additional one hour. Methanol (170 mL) is then added tostop the reaction. HPLC showed the presence of approximately 70%product. The solvent is evaporated and triturated with CH₃CN (200 mL).The residue is dissolved in CHCl₃ (1.5 L) and extracted with 2×500 mL ofsaturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phase isdried over Na₂SO₄, filtered and evaporated. 275 g of residue isobtained. The residue is purified on a 3.5 kg silica gel column, packedand eluted with EtOAc/hexane/acetone (5:5:1) containing 0.5% Et₃NH. Thepure fractions may be evaporated to give 164 g of product. Approximately20 g additional is obtained from the impure fractions to give a totalyield of 183 g (57%).

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M),DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) may becombined and stirred at room temperature for 24 hours. The reaction ismonitored by TLC by first quenching the TLC sample with the addition ofMeOH. Upon completion of the reaction, as judged by TLC, MeOH (50 mL) isadded and the mmixture evaporated at 35° C. The residue is dissolved inCHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers may be backextracted with 200 mL of CHCl₃. The combined organics may be dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue is purified on a 3.5 kg silica gel column andeluted using EtOAc/hexane (4:1). Pure product fractions may beevaporated to yield 96 g (84%). An additional 1.5 g is recovered fromlater fractions.

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

A first solution is prepared by dissolving3′-O-acetyl2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96 g,0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44 M)is added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L), cooledto −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃ isadded dropwise, over a 30 minute period, to the stirred solutionmaintained at 0-10° C., and the resulting mixture stirred for anadditional 2 hours. The first solution is added dropwise, over a 45minute period, to the latter solution. The resulting reaction mixture isstored overnight in a cold room. Salts may be filtered from the reactionmixture and the solution is evaporated. The residue is dissolved inEtOAc (1 L) and the insoluble solids may be removed by filtration. Thefiltrate is ished with 1×300 mL of NaHCO₃ and 2×300 mL of saturatedNaCl, dried over sodium sulfate and evaporated. The residue istriturated with EtOAc to give the title compound.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

A solution of3′-O-acetyl-2′-O-methoxyethyl-5′-Odimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) is stirred atroom temperature for 2 hours. The dioxane solution is evaporated and theresidue azeotroped with MeOH (2×200 mL). The residue is dissolved inMeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas is added and the vesselheated to 100° C. for 2 hours (TLC showed complete conversion). Thevessel contents may be evaporated to dryness and the residue isdissolved in EtOAc (500 mL) and washed once with saturated NaCl (200mL). The organics may be dried over sodium sulfate and the solvent isevaporated to give 85 g (95%) of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M)is dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M) isadded with stirring. After stirring for 3 hours, TLC showed the reactionto be approximately 95% complete. The solvent is evaporated and theresidue azeotroped with MeOH (200 mL). The residue is dissolved in CHCl₃(700 mL) and extracted with saturated NaHCO₃ (2×300 mL) and saturatedNaCl (2×300 mL), dried over MgSO₄ and evaporated to give a residue (96g). The residue is chromatographed on a 1.5 kg silica column usingEtOAc/hexane (1:1) containing 0.5% Et₃NH as the eluting solvent. Thepure product fractions may be evaporated to give 90 g (90%) of the titlecompound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) is dissolved in CH₂Cl₂ (1 L) Tetrazole diisopropylamine (7.1g) and 2-cyanoethoxy-tetra-(isopropyl) phosphite (40.5 mL, 0.123 M) maybe added with stirring, under a nitrogen atmosphere. The resultingmixture is stirred for 20 hours at room temperature (TLC showed thereaction to be 95% complete). The reaction mixture is extracted withsaturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueouswashes may be back-extracted with CH₂Cl₂ (300 mL), and the extracts maybe combined, dried over MgSO₄ and concentrated. The residue obtained ischromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1) asthe eluting solvent. The pure fractions may be combined to give 90.6 g(87%) of the title compound. 2′-O-(Aminooxyethyl) nucleoside amiditesand 2′-O-(dimethylaminooxyethyl) nucleoside amidites

2′-(Dimethylaminooxyethoxy) Nucleoside Amidites

2′-(Dimethylaminooxyethoxy) nucleoside amidites [also known in the artas 2′-O-(dimethylaminooxyethyl) nucleoside amidites] are prepared asdescribed in the following paragraphs. Adenosine, cytidine and guanosinenucleoside amidites are prepared similarly to the thymidine(5-methyluridine) except the exocyclic amines are protected with abenzoyl moiety in the case of adenosine and cytidine and with isobutyrylin the case of guanosine.

5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0 g,0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) maybe dissolved in dry pyridine (500 ml) at ambient temperature under anargon atmosphere and with mechanical stirring.tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol)is added in one portion. The reaction is stirred for 16 h at ambienttemperature. TLC (Rf 0.22, ethyl acetate) indicated a complete reaction.The solution is concentrated under reduced pressure to a thick oil. Thisis partitioned between dichloromethane (1 L) and saturated sodiumbicarbonate (2×1 L) and brine (1 L). The organic layer is dried oversodium sulfate and concentrated under reduced pressure to a thick oil.The oil is dissolved in a 1:1 mixture of ethyl acetate and ethyl ether(600 mL) and the solution is cooled to −10° C. The resulting crystallineproduct is collected by filtration, washed with ethyl ether (3×200 mL)and dried (40° C, 1 mm Hg, 24 h) to 149 g (74.8%) of white solid. TLCand NMR may be consistent with pure product.

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

In a 2 L stainless steel, unstirred pressure reactor is added borane intetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood and withmanual stirring, ethylene glycol (350 mL, excess) is added cautiously atfirst until the evolution of hydrogen gas subsided.5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) may be added with manualstirring. The reactor is sealed and heated in an oil bath until aninternal temperature of 160° C. is reached and then maintained for 16 h(pressure<100 psig). The reaction vessel is cooled to ambient andopened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T sideproduct, ethyl acetate) indicated about 70% conversion to the product.In order to avoid additional side product formation, the reaction isstopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warmwater bath (40-100° C.) with the more extreme conditions used to removethe ethylene glycol. [Alternatively, once the low boiling solvent isgone, the remaining solution can be partitioned between ethyl acetateand water. The product will be in the organic phase.] The residue ispurified by column chromatography (2 kg silica gel, ethylacetate-hexanes gradient 1:1 to 4:1). The appropriate fractions may becombined, stripped and dried to product as a white crisp foam (84 g,50%), contaminated starting material (17.4 g) and pure reusable startingmaterial 20 g. The yield based on starting material less pure recoveredstarting material is 58%. TLC and NMR may be consistent with 99% pureproduct.

2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) is mixed with triphenylphosphine (11.63 g, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It is then dried overP₂O₅ under high vacuum for two days at 40° C. The reaction mixture isflushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) isadded to get a clear solution. Diethylazodicarboxylate (6.98 mL, 44.36mmol) is added dropwise to the reaction mixture. The rate of addition ismaintained such that resulting deep red coloration is just dischargedbefore adding the next drop. After the addition is complete, thereaction is stirred for 4 hrs. By that time TLC showed the completion ofthe reaction (ethylacetate:hexane, 60:40). The solvent is evaporated invacuum. Residue obtained is placed on a flash column and eluted withethyl acetate:hexane (60:40), to get2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819 g, 86%).

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) is dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) is added dropwise at −10° C. to 0°C. After 1 h the mixture is filtered, the filtrate is washed with icecold CH₂Cl₂ and the combined organic phase is washed with water, brineand dried over anhydrous Na₂SO₄. The solution is concentrated to get2′-O-(aminooxyethyl) thymidine, which is then dissolved in MeOH (67.5mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) is addedand the resulting mixture is stirred for 1 h. Solvent is removed undervacuum; residue chromatographed to get5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridineas white foam (1.95 g, 78%).

5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) is dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride(0.39 g, 6.13 mmol) is added to this solution at 10° C. under inertatmosphere. The reaction mixture is stirred for 10 minutes at 10° C.After that the reaction vessel is removed from the ice bath and stirredat room temperature for 2 h, the reaction monitored by TLC (5% MeOH inCH₂Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) is added and extracted withethyl acetate (2×2O mL). Ethyl acetate phase is dried over anhydrousNa₂SO₄, evaporated to dryness. Residue is dissolved in a solution of 1MPPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL, 3.37 mmol) isadded and the reaction mixture is stirred at room temperature for 10minutes. Reaction mixture cooled to 10° C. in an ice bath, sodiumcyanoborohydride (0.39 g, 6.13 mmol) is added and reaction mixturestirred at 10° C. for 10 minutes. After 10 minutes, the reaction mixtureis removed from the ice bath and stirred at room temperature for 2 hrs.To the reaction mixture 5% NaHCO₃ (25 mL) solution is added andextracted with ethyl acetate (2×25 mL). Ethyl acetate layer is driedover anhydrous Na₂SO₄ and evaporated to dryness . The residue obtainedis purified by flash column chromatography and eluted with 5% MeOH inCH₂Cl₂ to get5-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6 g, 80%).

2′-O-(dimethylaminooxyethyl)-5-methyluridine

Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) is dissolved in dryTHF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH). Thismixture of triethylamine-2 HF is then added to5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reactionis monitored by TLC (5% MeOH in CH₂Cl₂). Solvent is removed under vacuumand the residue placed on a flash column and eluted with 10% MeOH inCH₂Cl₂ to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg,92.5%).

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) isdried over P₂O₅ under high vacuum overnight at 40° C. It is thenco-evaporated with anhydrous pyridine (20 mL). The residue obtained isdissolved in pyridine (11 mL) under argon atmosphere.4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytritylchloride (880 mg, 2.60 mmol) is added to the mixture and the reactionmixture is stirred at room temperature until all of the startingmaterial disappeared. Pyridine is removed under vacuum and the residuechromatographed and eluted with 10% MeOH in CH₂Cl₂ (containing a fewdrops of pyridine) to get5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.13 g, 80%).

5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67mmol) is co-evaporated with toluene (20 mL). To the residueN,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) is added and driedover P₂O₅ under high vacuum overnight at 40° C. Then the reactionmixture is dissolved in anhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N¹,N²-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) is added. The reaction mixture is stirred at ambient temperaturefor 4 hrs under inert atmosphere. The progress of the reaction ismonitored by TLC (hexane:ethyl acetate 1:1). The solvent is evaporated,then the residue is dissolved in ethyl acetate (70 mL) and washed with5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer is dried over anhydrousNa₂SO₄ and concentrated. Residue obtained is chromatographed (ethylacetate as eluent) to get5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]as a foam (1.04 g, 74.9%).

2′-(Aminooxyethoxy) nucleoside amidites 2′-(Aminooxyethoxy) nucleosideamidites [also known in the art as 2′-O-(aminooxyethyl) nucleosideamidites] are prepared as described in the following paragraphs.Adenosine, cytidine and thymidine nucleoside amidites are preparedsimilarly.N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

The 2′-O-aminooxyethyl guanosine analog may be obtained by selective2′-O-alkylation of diaminopurine riboside. Multigram quantities ofdiaminopurine riboside may be purchased from Schering AG (Berlin) toprovide 2′-O-(2-ethylacetyl)diaminopurine riboside along with a minoramount of the 3′-O-isomer. 2′-O-(2-ethylacetyl)diaminopurine ribosidemay be resolved and converted to 2′-O-(2-ethylacetyl)guanosine bytreatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D.,Guinosso, C. J., WO 94/02501 A1 940203.) Standard protection proceduresshould afford 2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosineand2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosinewhich may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine.As before the hydroxyl group may be displaced by N-hydroxyphthalimidevia a Mitsunobu reaction, and the protected nucleoside mayphosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,40-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

2′-dimethylaminoethoxyethoxy (2′-DMAEOE) Nucleoside Amidites

2′-dimethylaminoethoxyethoxy nucleoside amidites (also known in the artas 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂, or2′-DMAEOE nucleoside amidites) are prepared as follows. Other nucleosideamidites are prepared similarly.

2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl Uridine

2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) is slowlyadded to a solution of borane in tetrahydrofuran (1 M, 10 mL, 10 mmol)with stirring in a 100 mL bomb. Hydrogen gas evolves as the soliddissolves. O2-,2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodiumbicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oilbath and heated to 155 C. for 26 hours. The bomb is cooled to roomtemperature and opened. The crude solution is concentrated and theresidue partitioned between water (200 mL) and hexanes (200 mL). Theexcess phenol is extracted into the hexane layer. The aqueous layer isextracted with ethyl acetate (3×200 mL) and the combined organic layersare washed once with water, dried over anhydrous sodium sulfate andconcentrated. The residue is columned on silica gel usingmethanol/methylene chloride 1:20 (which has 2% triethylamine) as theeluent. As the column fractions are concentrated a colorless solid formswhich is collected to give the title compound as a white solid.

5′-O-dimethoxytrityl-2′-O-[2 (2-N,N-dimethylaminoethoxy)ethyl)]-5-methylUridine

To 0.5 g (1.3 mmol) of2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl uridine in anhydrouspyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride(DMT-Cl, 0.87 g, 2 eq.) are added and stirred for 1 hour. The reactionmixture is poured into water (200 mL) and extracted with CH2Cl2 (2×200mL). The combined CH2Cl2 layers are washed with saturated NaHCO3solution, followed by saturated NaCl solution and dried over anhydroussodium sulfate. Evaporation of the solvent followed by silica gelchromatography using MeOH:CH2Cl2:Et3N (20:1, v/v, with 1% triethylamine)gives the title compound.

5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

Diisopropylaminotetrazolide (0.6 g) and 2-cyanoethoxy-N,N-diisopropylphosphoramidite (1.1 mL, 2 eq.) are added to a solution of5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine(2.17 g, 3 mmol) dissolved in CH2Cl2 (20 mL) under an atmosphere ofargon. The reaction mixture is stirred overnight and the solventevaporated. The resulting residue is purified by silica gel flash columnchromatography with ethyl acetate as the eluent to give the titlecompound.

Example 2 Oligonucleotide Synthesis

Unsubstituted and substituted phosphodiester (P═O) oligonucleotides aresynthesized on an automated DNA synthesizer (Applied Biosystems model380B) using standard phosphoramidite chemistry with oxidation by iodine.

Phosphorothioates (P═S) are synthesized as for the phosphodiesteroligonucleotides except the standard oxidation bottle is replaced by 0.2M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile forthe stepwise thiation of the phosphite linkages. The thiation wait stepis increased to 68 sec and is followed by the capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (18 h), the oligonucleotides may be purified byprecipitating twice with 2.5 volumes of ethanol from a 0.5 M NaClsolution. Phosphinate oligonucleotides are prepared as described in U.S.Pat. No. 5,508,270, herein incorporated by reference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporatedby reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.No. , 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated byreference.

Alkylphosphonothioate oligonucleotides are prepared as described inpublished PCT applications PCT/US94/00902 and PCT/US93/06976 (publishedas WO 94/17093 and WO 94/02499, respectively), herein incorporated byreference.

3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Example 3 Oligonucleoside Synthesis

Methylenemethylimino linked oligonucleosides, also identified as MMIlinked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Example 4 Synthesis of Chimeric Oligonucleotides

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides can be of several different types.These include a first type wherein the “gap” segment of linkednucleosides is positioned between 5′ and 3′ “wing” segments of linkednucleosides and a second “open end” type wherein the “gap” segment islocated at either the 3′ or the 5′ terminus of the oligomeric compound.Oligonucleotides of the first type are also known in the art as“gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[2′-O—Me]--[2′-O-deoxy]--[2′-O—Me] Chimeric PhosphorothioateOligonucleotides

Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-O-deoxy phosphorothioate oligonucleotide segments are synthesizedusing an Applied Biosystems automated DNA synthesizer Model 380B, asabove. Oligonucleotides are synthesized using the automated synthesizerand 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portionand 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′wings. The standard synthesis cycle is modified by increasing the waitstep after the delivery of tetrazole and base to 600 s repeated fourtimes for RNA and twice for 2′-O-methyl. The fully protectedoligonucleotide is cleaved from the support and the phosphate group isdeprotected in 3:1 ammonia/ethanol at room temperature overnight thenlyophilized to dryness. Treatment in methanolic ammonia for 24 hrs atroom temperature is then done to deprotect all bases and sample is againlyophilized to dryness. The pellet is resuspended in 1M TBAF in THF for24 hrs at room temperature to deprotect the 2′ positions. The reactionis then quenched with 1M TEAA and the sample is then reduced to ½ volumeby rotovac before being desalted on a G25 size exclusion column. Theoligo recovered is then analyzed spectrophotometrically for yield andfor purity by capillary electrophoresis and by mass spectrometry.

[2′-O-(2-Methoxyethyl)]--[2′-deoxy]--[2′-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[2′-O-(2-methoxyethyl)]--[2′-deoxy]--[-2′-O-(methoxyethyl)] chimericphosphorothioate oligonucleotides may be prepared as per the procedureabove for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]--[2′-deoxyPhosphorothioate]--[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[2′-O-(2-methoxyethyl phosphodiester]--[2′-deoxyphosphorothioate]--[2′-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidizationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/oligonucleosides are synthesized according toU.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 5 Oligonucleotide Isolation

After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides or oligonucleosides are purified byprecipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol.Synthesized oligonucleotides may be analyzed by polyacrylamide gelelectrophoresis on denaturing gels and judged to be at least 85% fulllength material. The relative amounts of phosphorothioate andphosphodiester linkages obtained in synthesis may be periodicallychecked by ³¹P nuclear magnetic resonance spectroscopy, and for somestudies oligonucleotides may be purified by HPLC, as described by Chianget al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material may be similar to those obtained with non-HPLCpurified material.

Example 6 Alternative Pathway Reconstitution

The alternative pathway was reconstituted with purified human proteinsessentially as described by Keil (Keil et al., Am J Hematol1995;50(4):254-62).

Example 7 Complement Activity Assay

Measurement of complement activity was accomplished by measuring C3convertase activity by combining the following C3 (125 μg/ml), Factor B(20 μg/ml) and Factor D (0.2 μg/ml) in Hand's Balanced Salt Solution(HBSS) buffered with 5 mM HEPES, pH 7.2. Incubations may be carried outunder ambient conditions in the presence of oligonucleotide(concentrations up to 300 μg/ml) . Aliquots may be removed at selectedintervals and immediately diluted 50-fold in ice cold ELISA dilutionbuffer. Complement split products may be measured by ELISA.

In another example measurement of complement activity was accomplishedby measuring C3 convertase activity by combining the following C3 (125μg/ml), Factor B (20 μg/ml) Factor D (0.2 μg/ml), Factor H (25 μg/ml)and Factor I (2 μg/ml) in Hand's Balanced salt Solution (HBSS) bufferedwith 5 mM HEPES, pH 7.2. Incubations were carried out under ambientconditions in the presence of oligonucleotide concentrations (up to 300μg/ml). Aliquots were removed at selected intervals and immediatelydiluted 50-fold in ice cold ELISA dilution buffer. Complement splitproducts were measured by ELISA. Complement activation in serum wasmeasured in both rhesus monkey and human serum as follows:

Dilutions of oligonucleotides were added to normal human or rhesus serumat a 1:10-1:20 ratio, v/v. The samples were incubated at 37° C. andaliquots removed at selected intervals. Complement activation wasterminated by either placing the aliquots in an acid precipitatingreagent for RIA determinations, or by diluting the aliquots 1:50 in icecold sample diluent for ELISA determinations.

As controls for some experiments zymosan A (500 μg/ml) or cobra venomfactor (CVF; 2 U/ml) were used to activate the alternative pathway inthe presence of the oligonucleotide. Each was added at a final volume of1:20.

Example 8 In vivo Activation of Complement

Cynomolgus monkeys received single doses of 2 to 20 mg/kg ofoligonucleotide or a vehicle control solution by i.v. infusion forperiods ranging from 2 to 120 minutes. Measurement:

The level of complement split products Bb, C3a , C4a and C5a wasdetermined in EDTA plasma samples using commercially available (AmershamLife Sciences, Amersham, Little Chalfont, Buckinghamshire, England;Quidel, San Diego, Calif.) radioimmunoassay or enzyme-linkedimmunosorbent assay kits.

Total hemolytic complement activity in serum (CH50) was assayed in serumsamples using the standard hemolytic assay (Harbeck et al., DiagnosticImmunology Laboratory Manual. pp 9-20, Raven Press, New York, 1991).Factor H concentrations in monkey plasma was determined by radialimmunodiffusion (Harbeck et al.) using an anti-human Factor H antibody.

1 1 20 DNA Artificial Sequence Description of ArtificialSequenceSynthetic 1 gcccaagctg gcatccgtca 20

What is claimed is:
 1. A method for modulating complement activation ina cell, tissue or a bodily fluid comprising independently administeringto said cell, tissue or bodily fluid a first concentration and a second,independent concentration of an oligonucleotide which comprises one ormore phosphorothioate modifications, wherein said oligonucleotideinitiates complement activation at said first concentration and inhibitscomplement activation at said second, independent concentration.
 2. Themethod of claim 1, wherein said first concentration is less than orequal to 80 μg/ml.
 3. The method of claim 1, wherein said secondconcentration is at least 200 μg/ml.